Novel Ion Chromatographic Stationary Phases for the Analysis of Complex

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Novel ion chromatographic stationary phases for the analysis of complexmatrices

Brett Paull*a and Pavel N. Nesterenkob

Received 27th April 2004, Accepted 16th August 2004

First published as an Advance Article on the web 21st October 2004DOI: 10.1039/b406355b

Ion chromatography (IC) has a proven track record in the determination of inorganic and organic

anions and cations in complex matrices. Recently, application of IC to the separation and

determination of bio-molecules such as amino acids, carbohydrates, nucleotides, proteins and

peptides has also received much attention. The key to the determination of all of the above species

in the most analytically challenging complex matrices is the ability to manipulate selectivity

through control of stationary phase chemistry, mobile phase chemistry and the choice of detection

method. This Tutorial Review summarises some of the most significant recent advances made in

IC stationary phase technology. In particular, the review details stationary phases specifically

designed for ion analysis in complex sample matrices, and considers in which direction future

stationary phase development might proceed.

1 Recent reviews

Reviews detailing various advances in IC technology have been

fairly numerous in recent years and can be categorised as those

detailing general advances in IC as a whole1,2 and those which

focus on specific aspects of IC technology, such as new

stationary phases,3,4 advances in suppressor technology,5 and

advances in detection methods in general.6 Added to these

volumes we also have reviews of particular fields of IC

application, including drinking water,7 food stuffs,8 biological

liquids,9

saline solutions,10

the semi-conductor industry,11

environmental samples,

12–15

and last but not least, a reviewof sample treatment techniques and methodologies for IC.16

Finally, we can include a number of personal perspectives on

the historical progress of various aspects of IC17–20 and the

odd miscellaneous item such as a review of IC methods for

simultaneous separations of anions and cations.21 Obviously

this is only a selection of such reviews and many more can be

easily obtained, however the depth of material does act to

highlight the importance of the technique in the vast field of

inorganic analysis, and its status as the dominant method for

anion analysis in particular shows no sign of abating.

One area that still poses a significant challenge to the ion

chromatographer is the application of IC to complex sample

matrices. When using the phrase ‘complex matrix’ in this

context, one predominantly means solutions of high ionic

strength or samples containing large disparities between the

concentrations of the analyte ions and other species present

within the sample. We can also bracket biological solutions asso-called ‘complex matrices’, as they often contain high

concentrations of large bio-molecules such as peptides and

proteins and lower levels of small inorganic and organic ions.

For the sake of this review we will also classify non-aqueous

solutions as ‘complex matrices’. What we are not talking about

here are samples that simply require sample pretreatment,

such as those containing high levels of particulate matter or*[email protected]

Brett Paull

Brett Paull is a Lecturer and researcher within the School

of Chemical Sciences and

National Centre for SensorResearch, Dublin CityUniversity (DCU), Ireland.

Dr Paull obtained a PhD inanalytical chemistry from

Plymouth University, UK.Prior to joining DCU, hewas an Associate Lecturer

at the University of Tasmania, Australia. DrPaull’s research at DCU is

focussed on the varied field

of separation science, in parti-cular ion chromatography.

Pavel N. Nesterenko

Pavel N. Nesterenko is aProfessor within the

Chemistry Department of

Lomonosov Moscow StateUniversity, Moscow, RussianFederation. Prof. Nesterenko

obtained a PhD (1982) and DSc (1999) in analytical

chemistry from LomonosovMoscow State University. Hisresearch interests are in the

development and design of newstationary phases for variousseparation modes in chemical analysis.

i-SECTION: TUTORIAL REVIEW www.rsc.org/analyst | The Analyst

134 | Analyst , 2005, 130, 134–146 This journal is ß The Royal Society of Chemistry 2005



semi-solids for example, unless of course the pre-treatment

step results in a strongly acidic or basic or otherwise

concentrated extract.

The key to the success of all chromatographic techniques is

the ability to control and manipulate selectivity. In liquid

chromatography this includes the selectivity exhibited by the

stationary phase, the mobile phase and the type of detection

method employed. In reality, the analyst will carefully controlselectivity in each of the above areas, and it is the combination

of these ‘selectivities’ that results in the ability to analyse some

of the most complex samples. In IC the nature of the

stationary phase in particular plays a significant role in

controlling selectivity due to the large range of stationary

phase chemistries available, this being in contrast to reversed-

phase liquid chromatography, which is dominated by similar

octadecylsilica based stationary phases. Therefore, this review

will focus on the singular aspect of recent advances in

stationary phase technology for ion analysis, and the attempts

being made to improve the range of stationary phases available

to apply to these complex sample types. This involves both the

development of new stationary phase materials and columncapacities, and perhaps most importantly, the development of

new stationary phase chemistries.

2 Anion analysis

There is a significant range of commercial anion exchange

columns currently available, albeit produced by a small

number of manufacturers and based upon a limited range of

stationary phase technologies and chemistries. The main

players in the IC industry are the Dionex Corporation,

Metrohm AG and Alltech Associates Inc., and one or

two smaller specialist column manufacturers, such as

Transgenomic Inc. The majority of anion exchange columns

produced and/or supplied by the above companies are polymer

rather than silica based, these being substituted polystyrene

divinylbenzene resins (PS-DVB) (e.g. the Hamilton PRPX

range, the Metrohm Metrosep range and the Phenomenex Star

Ion A300 IC columns), or methacrylate based resins (e.g.

Alltech Allsep and Novasep ranges). The largest single

producer of polymer based IC stationary phases is the

Dionex Corporation (IonPac range of columns), who produce

ethylvinylbenzene divinylbenzene (EVB-DVB) based resins

with a variety of differing structural designs, including surface

functionalised, agglomerated and grafted resins. For most of

the above producers, the ion exchange functional group used

remains the standard strong quarternary ammonium group,with some weak anion exchangers based upon tertiary amine

groups. Table 1 shows some examples of new anion exchange

columns on the market, together with some of the complex

applications to which they have been applied.

2.1 New selectivity in commercial anion exchangers

The above mentioned IC companies are continuously

striving to explore and develop new selectivities to overcome

increasingly complex sample matrices. The Dionex

Corporation have in the past few years developed a number

of new products specifically designed for certain applica-

tions.22–24,55,56,147,148,150–155The company recently released the

so-called ‘hydroxide selective’ IonPac AS16 column, specifi-

cally for the determination of polarisable anions such as

perchlorate, iodide, thiocyanate and thiosulfate, using NaOH

or KOH only eluents. The resin itself is a high capacity

macroporous 9 mm diameter EVB-DVB substrate with a

surface coverage of 65 nm diameter latex particles functiona-

lised with very hydrophilic alkanol quarternary ammonium

groups. This results in a stationary phase that exhibits ‘ultra-low’ hydrophobicity, which Dionex describe as being

optimised for the determination of the above anions in

scrubber solutions, process streams, and brines. However,

the biggest application of this new column will be the

monitoring of ultra-trace levels of perchlorate in drinking

and ground waters, whereby the high capacity of the phase

allows for large sample injection whilst still maintaining

resolution of the analyte from excess matrix anions, and

the selectivity results in reduced run times and improved

peaks shapes for perchlorate compared to alternative

columns.23

A second hydroxide selective anion exchanger new to the

market is the Dionex IonPac AS19 column. This high capacityresin (160 meq column21), according to the manufacturer,

exhibits optimised selectivity for bromate and bromide, and is

therefore particularly applicable to the determination of

bromate in drinking water. The stationary phase itself is based

upon a hyper-branched anion-exchange condensation poly-

mer, electrostatically attached to a macro-porous surface

sulfonated EVB-DVB resin. The high capacity of the column

once again allows large sample volume injection (up to 500 mL),

with which bromate detection limits in drinking water samples

of approximately 1 mg L21 can be obtained with suppressed

conductivity detection.24

2.2 Adjustable-capacity anion exchangers

An interesting development in stationary phase technology,

which has direct implications for the analysis of complex

sample matrices, is the commercial availability of so-called

‘adjustable capacity anion exchangers’ (not an ideal name,

given that any weak anion exchanger has an ‘adjustable

capacity’). These anion exchangers are based upon immobi-

lised macrocyclic ligands (either coated or chemically bonded),

which exhibit varying selectivities towards anionic species

depending upon the nature of a central coordinated metal

cation. Changing the coordinated cation during a chromato-

graphic run results in a change in column capacity, resulting in

what is effectively a ‘capacity gradient’. A number of earlyinvestigations illustrated the potential advantages of this

approach,25–30

including short column regeneration time after

capacity gradient separations, low baseline drifts during

gradient runs and the use of eluents that are simple in

composition and low in ionic strength. The commercial

product that has evolved from these early studies is the

Dionex IonPac Cryptand A1 column, which is based on a

cryptand molecule, covalently attached to a macroporous,

EVB-DVB resin. A cryptand is a bi-cyclic compound capable

of complexing metal cations such as sodium, lithium and

potassium, thus forming the anion exchange site. Each metal

produces a specific capacity range, which is directly related to

This journal is ß The Royal Society of Chemistry 2005 Analyst , 2005, 130, 134–146 | 135



the metal–ligand binding constant. Several publications have

emerged recently which detail the use of this new anion

exchanger for the analysis of complex mixtures of inorganic

anions. For example, Woodruff et al. have utilised hydroxide

gradients, varying both hydroxide concentration and the

nature of the subsequent metal ion (LiOH, NaOH and

KOH), to control column capacity in order to initially retain,

and then rapidly elute, large concentrations of matrix

anions.31–33 Woodruff et al. applied this technique to the

determination of trace anions in 2% sulfuric acid,31

the

simultaneous determination of inorganic, organic and polari-

sable anions in industrial wastewater,32 and the determination

of chloride and sulfate in semi-conductor-grade etchants,

comprised of acetic acid, nitric acid and phosphoric acid.33

Fig. 1 shows the ion chromatogram of an alkaline (pH 14)

industrial wastewater sample from a light hydrocarbon plant,

run on a IonPac Cryptand A1 column using a KOH and LiOH

combined capacity and concentration gradient.

Table 1 New stationary phases designed for IC of inorganic species in complex matrices—anion exchangers

Stationary phaseBondedgroups

Column properties

Applications to analysis of complex matrices Ref.d p/mm

Columnsize/mm

Capacity/meqcolumn21 Matrix

IonPac AS9-HC –N+R2R9OH 9.0 250 6 4.0 190 Macroporous 200 nmpores; EVB-DVB,55%; 90 nm latex

beads with 15%cross-linking

Determination of trace anions inconcentrated weak acids withion-exclusion pretreatment,

organic solvents

56, 150

IonPac AS11-HC –N+R2R9OH 9.0 250 6 4.0 290 Macroporous 200 nmpores; EVB-DVB,55%; 70 nm latexbeads with 6%cross-linking

Determination of trace anions inmethanesulfonic and phosphoricacids with ion-exclusionpre-treatment. Determinationof ClO4

2 in fertilizers

55, 56, 151

IonPac AS15 –N+R2R9OH 8.5 250 6 2.0 56 EVB-DVB, 55%;10 nm pores

Determination of trace anions(CO3

22, Cl2, SO422 and PO4

32)in 7 g L21 solution of NaNO3

and CO322, Cl2 in 0.7% nitric

acid

148

IonPac AS16 –N+R2R9OH 5.0 250 6 2.0 MacroporousEVB-DVB, 55%;80 nm latexbeads with 1%

crosslinking

Simultaneous determination of F2, CO3

22, SO422, glycerol,

sorbitol, saccharin,monofluorophosphate, PO4

32,

pyrophosphate andtripolyphosphate in toothpaste

147

9.0 250 6 4.0 170 Determination of trace ClO42

(5 ppb) in drinking water inpresence of 200 ppm Cl2, infertilizers

23, 152, 153

IonPac AS17 –N+R2R9OH 10.5 250 6 4.0 30 MicroporousEVB-DVB, 55%;75 nm latex beadswith 6% crosslinking

The determination of trace levelphosphorus in purified quartz,trace anions in boric acid

154, 155

IonPac AS19 –N+R2R9OH 7.5 250 6 4.0 160 MacroporousEVB-DVB, 55%

The determination of tracebromate (5ppb) in drinkingwater

24

Novosep A-2 –N+R3 5.0 250 6 4.0 Polyvinylalcohol Determination of commoninorganic anions and oxyhalides(EPA Method 300.1)

Metrosep A Supp 2,

identical toTransgenomicAN1

–N+R2R9OH 8.0–10 2506 4.0 35 PS-DVB, 8 nm pores;

surface area415 m2 g21

Analysis of total fluoride content

in dentifrices

156

Metrosep A Supp 4 –N+R3 9.0 250 6 4.0 46 Polyvinylalcohol Suitable for all routine tasks inwater analysis

Metrosep A Supp 5,identical toShodex ICSI-50 4E

–N+R3 5.0 100 6 4.0 39 Polyvinylalcohol Determination of trace level ClO42

by IC-MS157

Metrosep A Supp 7 –N+R3 5.0 250 6 4.0 — Polyvinylalcohol Determination of waterdisinfectant by-products,bromate in particular

IonPac CryptandA1

2,2,1 cryptand 5.0 150 6 3.0 73a Macroporous, 100 nmpores; EVB-DVB,45:55%

Determination of polyvalentanions including polyphosphatesand polysulfonates;alkanesulfonic acids in achromic acid plating bath

31, 32, 149

a Variable capacity depending on eluents used.




2.3 Zwitterionic stationary phases

The potential advantages to be gained from the use of

zwitterionic stationary phases in assorted modes of LC are

now reasonably well understood.34 Neutral hydrophilic

stationary phases show increased retention for polar and

hydrophilic compounds relative to reversed-phase substrates.

Recently we have seen the commercial availability of a silica

based covalently bonded zwitterionic column, the SeQuant

ZIC-HILIC column, especially designed for so-called ‘hydro-

philic interaction chromatography’ of nucleotides, amino acids

amines, phenols, carbohydrates and other polar analytes.35

This product came directly off the back of the work of Jiangand Irgum, who covalently functionalised both polymer36,37

and silica substrates38 with zwitterionic functional groups,

and showed how these phases could then be applied to the

simultaneous separation of inorganic anions and cations.

The use of coated zwitterionic phases for the separation of

inorganic and organic ions, (these being reversed-phase

substrates coated with zwitterionic surfactants) was first

proposed by Hu et al. in the early 1990’s.39 Since that time,

the technique, which was christened at the time ‘electrostatic

ion chromatography’ (EIC), has been extensively investigated

and applied to the analysis of many complex samples,

particularly biological fluids and seawater matrices.40–48 A

short review on the technique was published in 1998.49

Due tothe nature of the zwitterionic phases developed, ions within a

sample experience both attractive and repulsive electrostatic

forces as they travel through the stationary phase. The

positioning and nature of the anionic and cationic groups

within the immobilised zwitterion governs the relative strength

of these forces, which determines whether the stationary phase

shows a general selectivity towards anionic or cationic species.

Recently, Cook et al. considered a detailed retention mechan-

ism for this mode of IC, which proposed that the terminal

ionic group (in this study sulfonate) of the adsorbed zwitterion

forms a charged layer on the surface of the stationary phase,

which acts as a Donnan membrane. The effective charge on

this membrane is then dependent upon the nature of the

oppositely charged eluent cations, which in turn affects the

ability of analyte anions to pass through and interact with

the inner ionic site (in this case quarternary ammonium).50

Interestingly, it has been shown that with certain zwitter-

ionic stationary phases the effect of eluent concentration has a

much smaller effect upon analyte retention than simple ion

exchange,

42

and in certain configurations it has been shownthat the interaction of the analyte ions with the zwitterionic

phase increased with an increase in eluent concentration (up

to a certain concentration, after which retention remains

constant). This effect, together with a low affinity for sulfate

and chloride ions shown by most of the zwitterionic

surfactants investigated (see Fig. 2(a) for a Zwittergent 3–14

coated column), means the direct analysis of high ionic

strength samples, particularly highly saline samples is possible

using this approach. Specific complex applications demon-

strated using EIC include the direct determination of UV

absorbing anions in biological fluid including serum, urine

and saliva,39,40 the determination of UV-absorbing anions

(nitrite, bromide, iodide and nitrate) in seawater,42,43

therapid determination of iodide in seawater,45 and the determi-

nation of iodide in iodised table salt.46 Fig. 2 shows the

determination of iodide in solutions of iodised table salt

containing 20 g L21 of NaCl using a zwitterion coated ODS

column.

2.4 New phases for organic acids

Usually organic acids can be separated by either ion-exclusion

on strong cation-exchange columns, as recently reviewed by

Fischer for environmental samples,51 or by ion exchange on

Fig. 2 Chromatograms showing; (a) the standard separation of

sulfate, chloride, nitrite, nitrate, chlorate, iodide and thiocyanate. (b)

A sample of iodised table salt (20 g L21) spiked with between 0.8 and

8 mM iodide. Column 5 ODS coated with zwittergent 3–14. Eluent 5

2 mM zwittergent 3–14. Reproduced with permission from ref. 46.

Fig. 1 Alkaline (pH 14) industrial wastewater from light hydrocar-

bon plant. Column 5 IonPac Cryptand A1 150 mm 6 3 mm id, 5 mm.

Eluent 5 10 mM KOH, at 5 min step to 22 mM LiOH, at 10 min step

to 40 mM LiOH. Flow rate 5 0.5 mL min21. Column temp. 5 35 uC.

Injection vol. 5 5 mL. Peaks 5 1—fluoride, 2—acetate, 3—formate,

4—propionate, 5—chloride, 6—carbonate, 7—sulfate, 8—thiosulfate.

Reproduced with permission from ref. 32.




anion exchange columns, as reviewed earlier by Hajos and

Nagy.52 For the improved separation of aliphatic organic

acids and alcohols in complex or high-ionic strength samples,

Dionex have recently designed the new moderately hydro-

phobic mixed sulfo/carboxylic acid functionalised IonPac

ICE-AS6 ion-exclusion column. This column has been

applied to the determination of a wide range of carboxylic

acids in fruit juice, landfill and composting and fermentationplant effluents53,54

and in combination with a Dionex AS11-

HC anion-exchange column, was found useful for the

determination of chloride, sulfate and nitrate in concentrated

phosphoric acid, semiconductor grade 49% HF and 70%

glycolic acids.55,56

3 Cation analysis

Although IC, as a technique for cation analysis of relatively

simple sample matrices, competes with alternative technolo-

gies, such as atomic absorption spectroscopy, it still remains a

potent analytical technique when it comes to the analysis of

complex sample matrices (which can cause substantial inter-

ference problems with alternative spectroscopic and electro-

chemical based techniques).

In cation exchange chromatography there are two main

approaches that have been taken in the development of

stationary phases for complex sample analyses. These are, (i)

the development of high capacity phases which can handle

concentrated samples and disproportionate concentration

ratios of analytes, and (ii) the alteration of selectivity by

incorporation of some form of selective analyte complexation

within the stationary phase. In the latter case, what results isessentially a ‘dual retention mechanism’ which gives the

analyst added scope to selectively manipulate the retention

of target analytes through control of various eluent conditions,

such as eluent pH, and not just eluent concentration. Table 2

shows some examples of cation exchange columns currently

available, which have been developed for complex sample

analysis.

3.1 Grafted moderate and high capacity cation exchangers

Recent years have seen the Dionex Corporation extend their

range of cation columns to include two new carboxylate

grafted cation exchangers. These are the IonPac CS16 and

CS17 columns, which are of high (8.4 meq column21) and

moderate capacity (1.45 meq column21), respectively. The

Table 2 New stationary phases designed for IC of inorganic species in complex matrices—cation exchangers

Stationary phase Bonded groups

Column properties


Columnsize/mm

Capacity/meqcolumn21 Matrix

IonPac CS12A –COOH 8.5 250 6 4.0 2800 EVB-DVB,55%; 15 nmpores; 450 m2 g21

Trace-level quantificationof NH4

+ (50 ppb) inNaCl brine (1000:1 ratio)

67 –PO3H2 5.0 150 6 3.0 940

IonPac CS15 –COOH 8.5 250 6 4.0 2800 EVB-DVB,55%; 15 nmpores; 450 m2 g21

Determination of trace-levelNa+ (10 ppb) in amine-treated(100 ppm NH4

+) coolingwaters, trace-level NH4

+

(25 ppb) in environmentalwaste water containing a highNa+ concentration (100 ppm),trace level NH4

+ (10 ppb) ina KCl (100 ppm) soil extract

62, 63 –PO3H2

–18-crown-6 ether

IonPac CS16 –COOH 5.5 250 6 5.0 8400 EVB-DVB,55%; 15 nmpores; 450 m2 g21

Determination of trace levelNH4

+ (10 ppb) in highconcentrations of Na+

(100 ppm); trace level Na+

(10 ppb) in high concentrationsof NH4

+ or amines (100 ppm),alkali and alkaline-earth metalions in acid samples (up to0.1M) without pH adjustment

58, 59

IonPac CS17 –COOH 7.0 250 6 5.0 1450 EVB-DVB,

55%; 15 nmpores; 450 m2 g21

Simultaneous separation of alkali,

alkaline-earth cations andboiler water amine additiveswith gradient elution.

57

Universal cation –COOH 3.0 or 7.0 100 6 4.6 Silica based Determination of cations at tracelevels in ice core samples

158

Metrosep C 2 –COOH 5.0 250 6 4.0 194 Silica based Determination of 7 mg L21 sodiumin presence of 7 mg L21

monoethanolamine in boilerwater

LiChrosil IC CA –COOH 5.0 100 6 4.6 Silica coatedwith polymer

Simultaneous separation of alkali,alkaline-earth, transition metalcations, ammonia

Hamilton PRP-X800 –COOH 5.0 150 6 4.0 3700 meq g21 PS-DVB,macroporous

Trace alkaline-earth and transitionmetals in brines

70

Ammonia isotopecolumn

–SO32 5.0 300 6 4.0 PS-DVB, 12% Determination of 14NH4

+ and15NH4

+ ion ratios in sea water71




grafting technology used to produce the above resins results in

a hydrophilic surface, which shields the hydrophobic EVB-

DVB core. This is achieved through first modifying the resin

with a non-functional coating and subsequently grafting a

polymeric ion exchanger to this coating. With the CS17

column, this results in improved peak shape for hydrophobic

cations, without the need for the addition of organic

solvents.

57

This column has been applied to the simultaneousseparation of Group I and II cations, ammonium and

ethanolamines in boiler water, using a simple methanesulfonic

acid gradient.57

The higher capacity CS16 column is particularly suited to

the analysis of samples with large variations in cation content.

By simply utilising its high capacity the column has been used

for the determination of trace ammonium ions in excess of

sodium (10 mg L21 ammonium in 100 mg L21 sodium)58 and

has also been applied to the determination of trace ammonium

(0.22 mg L21) in industrial wastewater.59 The column is also

suitable for the analysis of acidic samples, containing up to

100 mM hydronium ion, and as such has also been applied to

the determination of Group I and II cations in acidic soilextracts.59

3.2 Cation exchangers incorporating crown ethers

The incorporation of crown ethers into eluents for cation

exchange chromatography to improve resolution of closely

eluting metal ions is well established and has been shown

again recently by Ohta et al., who used crown ether containing

eluents with silica gel columns for the simultaneous separation

of Group I and II cations.60 For obvious reasons bonding

crown ethers to the stationary phase itself is a more complex

procedure, however, many workers have produced such

phases, a review of which has been compiled by Lamband Smith.61

A commercial end-product of this early research is the

Dionex IonPac CS15 column, which is a polymeric macro-

porous resin functionalised with carboxylic acid groups,

phosphonic acid groups and 18-crown-6 ether. The selectivity

exhibited through the combination of the above functional

groups sees greater resolution of sodium and ammonium

than shown with alternative cation exchangers and also

sees potassium elute after calcium and magnesium.

Obviously, this selectivity is ideal for the analysis of samples

with excessively high concentrations of one or more of

these cations, and this has been notably demonstrated

with the resolution of potassium and sodium62

and sodiumand ammonium,63 both at concentration ratios of 10,000:1,

on the IonPac CS15 column. Fig. 3 shows the determination

of sodium, potassium, calcium and magnesium in an

ammonium acetate extract of a soil sample, obtained using

the above column.

Crown ether containing stationary phases have also become

available through a number of other specialist manufacturers.

Although mainly developed for enantioselective separations

of amino acids, companies such as USmac Corporation

are now selling a silica based Opticrown range of columns

(http://www.opticrown.com/products.html),64 and most

recently, Alltech Associates have published results of work

investigating a 18-crown-6 bonded silica column which was

used in series with their Universal Cation column, allowing theisocratic separation of sodium and ammonium at a concentra-

tion ratio of 5000:1.65

3.3 Novel weak acid cation exchangers

The use of alternative functional groups to the standard

sulfonated and carboxylated substrates, will naturally result in

alternative stationary phase selectivities towards various

cations. The above mentioned IonPac CS15 column with

its mixed functional bed was a further development of the

IonPac CS12A column, which also had a mixed carboxylate/

phosphonate functionality. The incorporation of the complex-

ing phosphonate group improved resolution of certain metalions, in particular allowing the isocratic separation of Group I

and II cations together with manganese in under 12 min. 66 The

combination of this novel selectivity and the moderate/high

capacity of the CS12A column have seen it applied to such

complex challenges as the determination of trace calcium and

magnesium in 30% sodium chloride brines.67 Interestingly, the

complexing ability of the phosphonate groups within the

CS12A column has been shown to result in unusual selectivity

towards transition and heavy metal ions, with a strong affinity

in particular for bismuth and the uranyl ion under acidic

conditions.68,69

Recently, the Hamilton Company have also released a weak

acid cation exchanger with complexing capabilities. The PRP-X800 resin is PS-DVB based and functionalized with itaconic

acid. Its intended application is the simultaneous separation of

Group I and II cations, but due to the complexing nature of

itaconic acid, the column also shows strong affinity for

transition and heavy metals, particularly lead and copper.70

Under non-acidic eluent conditions, complexation exceeds

simple ion exchange as the dominant retention mechanism for

divalent metal ions, allowing potential application of this

column to higher ionic strength samples. This potential was

exploited recently with the determination of low mg L21

concentrations of magnesium, calcium, strontium and barium

in a 2.3 g L21 sodium chloride solution.70

Fig. 3 Chromatograms showing an ammonium acetate extract of

a soil sample. Column 5 IonPac CS15. Eluent 5 5 mM sulfuric acid,

9% acetonitrile. Column temp. 5 40 uC. Injection vol. 5 25 mL.

Peaks 5 1—sodium, 2—ammonium, 3—magnesium, 4—calcium,

5—potassium. Reproduced with permission from ref. 62.




3.4 Chelating stationary phases

When discussing the application of chelating stationary phases

for cation determinations using IC, it is important to

distinguish between those used simply for preconcentration

and matrix elimination purposes, and those employed for

actual high-performance analyte separations. The incorpora-

tion of short chelating columns within a modified IC system,

for on-line analyte preconcentration and removal from

complex matrices, prior to separation using ion exchange, is

the basis of the Dionex chelation ion chromatography (CIC)

system. This system has been successfully applied to theanalysis of many complex samples over the past 14 years since

its inception,72–84 including trace metals in seawater,72–74,77,83

geological digests,76,78–80 biological samples,72,80,82,84 drinking

water,81 fertilizers79 and reagent grade chemicals.82

However, here we will briefly focus on the application of

chelating stationary phases for the direct analysis of complex

sample matrices, so-called high-performance chelation ion

chromatography (HPCIC), as last reviewed by Jones and

Nesterenko in 1997.85 To-date the majority of such applica-

tions have been carried out using iminodiacetic acid (IDA)

modified polymer and silica based substrates, either covalently

bonded or dynamically coated onto the surface. The use of

IDA-silica phases is particularly well documented,86–100 pre-dominantly due to the fact that IDA-silica columns are

commercially available (Diasorb IDA, BioChemMack ST,

Moscow, Russia). This column shows strong selectivity

towards both alkaline earth metals and transition/heavy metal

metals over alkali metal ions at eluent pHs greater than y4–5

and y2–3, respectively, and therefore the selectivity is ideally

suited to the determination of these metals within samples

containing high levels of alkali salts, such as seawater and

industrial brines. Application of the IDA-silica column to the

analysis of complex sample matrices include cobalt, zinc and

cadmium spiked into seawater,86 zinc and lead in wastewater

from a galvanic bath,87 beryllium in acidic rock digest,88 trace

beryllium in natural waters and simulated seawater96 and trace

alkaline earth metals in NaCl and KCl brines.97 Alternative

covalently bonded chelating stationary phases investigated

recently include aminophosphonate functionalised silica,

which has also been applied to the determination of trace

beryllium, on this occasion in stream sediments.101,102 There

have also been a number of polymer based chelating phases

developed, such as a N -methyl-c-aminobutyrohydroxamate

resin, which was used for the determination of lanthanide

metal ions in seawater,77 and a nitrilotriacetate PS-DVB resin

used for the determination of transition/heavy metal ionsincluding uranium in seawater (detection with ICP-MS).103

Fourteen REE can be isocratically separated on a IDA-silica

column92,93 or on a methacrylate gel with attached 1-amino-

benzyl-1,2-diaminopropane-N ,N ,N 9,N 9-tetraacetic acid, except

for Eu and Gd which are co-eluted.104 Table 3 details

properties and some complex applications of chelating ion

exchange columns.

Simply coating (either permanently or dynamically)

reversed-phase substrates with hydrophobic chelating ligands

is an alternative, and perhaps simpler, approach to covalent

bonding. This approach has been extensively explored by

Jones and co-workers105–116 using polymeric substrates

coated with chelating dyestuffs (several containing one ormore IDA groups) and various heterocyclic carboxylic acids.

Within this substantial body of work, these coated substrates

have been applied to alkaline earth and transition/heavy

metals in concentrated salt solutions,106 transition/heavy

metals in coastal seawater,107 barium and strontium in excess

calcium containing matrices such as milk powder,108 trace

aluminium in seawater,109 traces of bismuth in lead metal,110

trace uranium in environmental waters,113 actinides in

environmental and biological samples,114 and the determina-

tion of trace metals in highly mineralised waters.116 Fig. 4

shows an application of this approach, using a dipicolinic

acid dynamically coated polymeric reversed-phase column for

Table 3 New stationary phases designed for IC of inorganic species in complex matrices—chelating ion exchangers

Stationaryphase Bonded groups

Column propertiesApplications toanalysis of complexmatrices Ref.d p/mm

Columnsize/mm Capacity/meq g21 Matrix

Diasorb IDA –N(CH2COOH)2 7.0 250 6 4.0 130 Modified silica, 13 nm Determination of tracealkaline-earth metalsin brines, seawater

96, 97

APAS –NHCH2PO3H2 5.0 250 6 4.0 100 Modified silica, 10 nm Determination of beryllium in astream sediment

101, 102

N -methyl P13 N -methyl-c-amino-butyro-hydroxamate

63–150 250 6 3.0 1.5–1.9 mmol g21 Poly(acrylonitrile), 8% Determination of lanthanides inseawater

77

NTA-resin Nitrilotriacetate 10 50 6 4.6 60 mmol g21 PS-DVB, 60% Determination of tracemetals (Co, Ni, Cu,Zn, Cd, Mo, Sb, Pb,U) in sea-water at1 mg L21 level byICP-MS interfacedwith an IC system

103

PDTA-resin 1-Aminobenzyl-1,2-diaminopropane-N,N, N 9,N 9-tetraacetic

acid

10 125 6 4.6 127 Macroporousglycidylmethacrylate gel

Isocratic separation of fourteen REE

104




the separation of actinide species within the acidic digest of

a human lung tissue reference material. The selectivity of

the chelating phase combined with the selective detection

method of high-resolution inductively coupled plasma

mass spectrometry (HR-ICP-MS) proved a very powerful

combination.

Table 4 shows details of some miscellaneous stationary

phases, which have been applied to complex analytical tasks.

This table includes novel phases that have been used for

simultaneous anion and cation separations.

4 New phases for bio-analysis

In addition to the need for robust IC methods for the

determination of small ions in biological matrices,121 there is

now also a strong growth in interest from IC manufacturers in

the application of IC systems to the sensitive and selective

determination of low and moderate molecular weight bio-

molecules, particularly those that are charged species under IC

separation conditions. In terms of the types of biological

samples being analysed for the presence of these bio-molecules,

it is safe to say practically all can be considered as ‘complex’

due to the very large number and varied nature of species

present. The list of bio-molecules, which can be readily

separated through ion-exchange and, hence determined using

IC, includes organic acids, amino acids, amino sugars,

nucleotides and carbohydrates. Of course, corresponding

oligomeric molecules, such as peptides, oligonucleotides and

oligosaccharides, can also be efficiently separated using ion

exchange stationary phases, although often additional reten-

tion effects are observed such as hydrophobic interactions and

size-exclusion, which can detrimentally affect peak shapes.Table 5 shows a range of new stationary phases designed for

bioanalytical applications of IC.

4.1 Amino acid columns

For many years the standard practice for amino acid analysis

was based upon cation exchange chromatography with a step

gradient of eluent concentration and pH, combined with

ninhydrine post-column reaction. More recently, an alternative

approach proposed by Dionex involves an anion-exchange

based separation, with electrolytic eluent generation of an

hydroxide gradient and detection using pulsed amperome-

try.122 The novel polymeric pellicular anion exchange resin

specifically developed for this, is commercially available as the

AminoPac PA10 column, and has been applied to amino acid

analysis of cell culture media and fermentation broths.122–124

Fig. 4 Chromatograms showing the separation of 239

Pu fromuranium, and consequently the 238U1H+ interference in NIST 4351

human lung reference material. Column 5 100 mm Polymer

Laboratories PLRP-S coated with dipicolinic acid. Eluent 5 0.1 mM

dipicolinic acid and 0.75 M HNO3. Detection 5 HR-ICP-MS.

Reproduced with permission from ref. 114.

Table 4 New stationary phases designed for IC of inorganic species in complex matrices—miscellaneous ion exchangers

Stationaryphase Bonded groups

Column properties


Columnsize/mm Capacity Matrix

ZIC-HILIC –N+(CH3)2(CH2)3SO32 5.0 Silica, 20 nm

poresDetermination of nucleotides,

amino acids, amines,phenols, carbohydrates,peptides, glucuronated/

glycosylated andphosphorylated compounds

35

PolyCat A Poly(aspartic)acid 5.0 200 6 4.6 Silica, 6 nmpores

Simultaneous determinationof inorganic anions (Cl2,NO3

2) and cations (Na+,K+, Mg2+, and Ca2+ in water

117, 118

TSKgelSuperIC-A/C

–COOH 3.0–4.0 1506 6.0 0.2 meq mL21 Polymethacrylate Improved version of TosohTSKgel OApak-A designedfor simultaneousdetermination of inorganicanions (SO4

22, Cl2, NO32)

and cations (Na+, NH4+,

K+, Mg2+, and Ca2+ in acidrain water

119

IonPacICE-Borate

–SO32 7.5 250 6 9.0 27 meq column21 Microporous,

DVB, 8%Trace borate analysis in

deionized water120




Table 5 New stationary phases designed for bioanalytical applications of IC (including organic acids)

Stationaryphase

Bondedgroups

d p/mm Dpore/nm

Capacity,column/mm Matrix Application Ref.

Organic acidsIonPac

ICE-AS6Mixed –SO3

2

and –COOHgroups

8.0 Microporous 27 meq column21,2506 9.0 mm

PS-acrylate-DVB,8% crosslinking,intermediatehydrophobic resin

Ion-exclusion determinationof organic acids in fruit juice, effluents of landfills,composting andfermentation plants

53, 54

Tosoh TSKgelOApak-A

–COOH 5.0 — 0.1 meq mL21,1506 7.8 mm

Polymethacrylate Vacancy chromatographyof aliphatic carboxylic acids

126

Shim-packIC-A3

–N+R3 — — 150 6 4.6 mm Hydrophilic resin Formate in methanogenicdegradation of butyrate

130

Amino acidsAminoPac

PA 10 –N+R3 5.0 250 6 2.0 mm EVB-DVB substrate

55% crosslinkingagglomerated with80 nm functionalizedlatex with 1%crosslinking

Analysis of proteinhydrolysates and complexmammalian cell cultures

122, 123

Determination of amino acidsin samples with high contentof carbohydrates

124

CarbohydratesESA

Sucrebead I

–N+R3 7.0 N/a 250 6 2.0 mm PS-DVB substrate Mono and disaccharides with

amperometric detectionMetroSep

Carb 1 –N+R3 5.0 1530 meq,

2506 4.6 mmPS-DVB substrate Separation of polysaccharides

with length of up to 30glucose units

127

CarboPacPA-20

–N+R3

difunctional6.5 ,1.0 nm 65 meq,

1506 3.0 mmEVB-DVB substrate

55% crosslinkingagglomerated with130 nm functionalisedlatex with 6%crosslinking

Fast separation of monosaccharides anddisaccharides, glycoproteins

128, 129

CarboPacPA-200

–N+R3 5.5 Microporous 90 meq,2506 3.0 mm

EVB-DVB substrate55% crosslinkingagglomerated with34 nm functionalizedlatex with 6%crosslinking

Separation of oligosaccharidesbased on fine structuraldifferences

Dynamically

loadedEVB-DVBresins

Decyl-2,2,2

cryptand

Various types of EVB-DVB substrates Mono-, di- and oligosaccharides 29

NucleotidesShodex IEC

DNApakDEAE 2.5 Non-porous 0.70 meq g21,

50 6 6.0 mmPolyhydroxymetha-

crylateSeparation of oligonucleotides

HydrocellQA NP10

–N+R3 Non-porous PS-DVB beads High speed analysis of smallnucleotides, oligonucleotidesand DNA fragments

DNAPacPA100

–N+R3 13 Non-porous 250 6 4.0 mm EVB-DVB substrate55% crosslinkingagglomerated with100 nm functionalizedlatex with 5%crosslinking

Resolution of oligonucleotidesup to 60 bases,oligonucleotides withsecondary structures,analysis phosphorothioate-based clinical samples

131

HydrocellNS 1500 –N+

R3 10 50 1506

2.1 mm Highly cross-linkedPS-DVB Separation of nucleotides from2 to 40 residues and DNArestriction fragments of upto 1000 base pairs

Zorbax Oligo Mixedhydrophobicand ion-exchange

5.0 15 125 6 4.0 mm Silica stabilized withzirconia layer,140 m2 g21

Oligonucleotides from 3 to48 residues

Primesep 200 Alkylsilica withembeddedCOOHgroups

5.0 10 250 6 4.6 mm Silica Separation of nucleosides 132

Proteins and peptidesBioBasic AX Polyethylene-

imine5.0 30 0.22 meq g21,

3006 4.6 mmSilica, 100 m2 g21 Separations of peptides,

proteins, nucleic acids




Fig. 5 shows the impressive separation of amino acids and

selected carbohydrates possible with the AminoPac column in

a diluted fermentation broth sample. In the application shown

a novel bi-modal integrated amperometric detection method

was used to improve detector selectivity. The two chromato-

grams shown are of the same sample, determined under twodiffering detector modes.

4.2 Carbohydrate columns

There has also been significant progress in the development of

new anion-exchangers designed for the separation of carbohy-

drates. As with amino acid analysis, the IC determination of

carbohydrates can be achieved through ion-exchange chroma-

tography, following by amperometric detection. The CarboPac

range of new columns from Dionex (Carbo-Pac PA20, and

PA200) offers a range of capacities and resin structures to suit

particular applications. The CarboPac PA200 is the newest of

the range, designed for mono- and disaccharide determina-

tions. The column is based upon a 5.5 mm EVB-DVB agglo-

merated resin and is the recommended column for separation

of oligosaccharides having fine structural differences such as

the composition and the sequence of the oligo-saccharides,

linkage isomerism, degree of sialylation, and degree of

branching. Examples of complex applications of these

CarboPac phases include the analysis of starch and the

determination of complex novel food carbohydrates125 and

glycoprotein monosaccharides.128,129 Methrom have also

moved into the bio-analysis market with the release of theirMetrosep Carb 1 carbohydrate column range, which are low

and medium capacity polystyrene based anion exchangers,

designed for the separation of mono- and disaccharides,

although also suitable for the separation of poly- and

oligosaccharides. Example applications include the carbohy-

drate analysis of vodka, amino aid solutions and plant

extracts. Recently, one more company Shiseido introduced

ESA Sucrebead I anion-exchange column for carbohydrate

determination with amperometric detection.

4.3 Nucleotides and oligonucleosides

Two types of anion-exchanger are seen to be suitable for the

separation of nucleotides, these being small particle size non-

porous substrates, such as those found in the Shodex IEC

DNApak and Hydrocell QA NP10 columns, and alternatively

larger porous particles, like those within the Hydrocell NS

1500 column (both from BioChrom Labs). The DNAPac PA-

100 column produced by Dionex is a pellicular agglomerated

anion exchange resin specifically designed to obtain resolution

of synthetic oligonucleosides to 60 bases and beyond, and the

resin based column is also compatible with strongly denaturing

conditions such as high pH and temperature conditions.131

Taking into account the zwitterionic nature of nucleotides and

oligonucleosides, various interactions and their combinations

can be realised for chromatographic resolution. Both cation-

and anion-exchangers,131 zwitterionic ion-exchangers,35 mixed

mode ion-exchange and reversed-phase bonded phases like

Zorbax Oligo produced by Agilent, or alkylsilica with

embedded ion-exchange groups like Primesep 200132 were

found useful for separation of these charged molecules.

4.4 Proteins and peptides

Ion-exchange chromatography has seen significant growth in

interest for use in the multidimensional chromatographic

fractionation of complex protein digests in proteomics studies.

Although polymeric ion-exchangers have long been used for

protein separations, in the past they could not compete with

ProPacSAX-10,WAX-10,WCX-10,SCX-10

–N+R3, –NR2, –COOH, –SO3

2

10 ,1.0 250 6 4.0 mm EVB-DVB, 55%crosslinking

High resolution of proteins withsmall differences in charge

OtherAlkion

PickeringLaboratories

–SO32 10 Non-porous Low capacity PS-DVB substrate Determination of biogenic and

polyamines in foods, beveragesand medicines, aminoglycosides,antibiotics in feeds, amine-containing herbicides

Table 5 New stationary phases designed for bioanalytical applications of IC (including organic acids) ( continued )

Stationaryphase

Bondedgroups

d p/mm Dpore/nm

Capacity,column/mm Matrix Application Ref.

Fig. 5 Chromatograms showing the separation of amino acids and

carbohydrates within a fermentation sample (diluted 1:250).

Column 5 Dionex AminoPac PA10. Eluent 5 NaOH gradient.

Detection5 Bi-modal integrated amperometric detection. Reproduced

with permission from ref. 122.




the resolution capabilities of reversed-phase sorbents.

Recently, specialist protein columns based on pellicular ion-

exchangers with a hydrophilic charged outer layer have been

introduced (ProPac series from Dionex), which offer much

better separation performance than traditional columns. For

example, a number of different C-termini Lys variants have

been separated and collected using the ProPac WCX-10

column.

133

Other stationary phases designed for proteinseparations are based upon the introduction of different ion-

exchange groups in alkyl chains, this approach has been used

in many new silica based phases, like for example, Kaseisorb

ODS-SAX or Primesep 200.132

5 Future directions

At this late stage of this review it seems appropriate to touch

on some specific recent directions in IC stationary phase

technology which are attracting much attention and which

show potential for the analysis of complex matrices.

5.1 Monolithic phases

Monolithic polymer and silica based anion and cation

exchangers are beginning to become available commercially

and will no doubt gain in popularity over the next few years.

On the polymer side a number of manufacturers are now

producing ion exchange functionalised short monolithic

columns for fast separations of bio-molecules from biological

matrices (Isco Swift columns and BIA Separations Convective

Interaction Media or CIM disks).134

There have also been

work carried out on zwitterionic functionalised polymer

monolithic columns, again primarily designed for the rapid

separation of large bio-molecules such as proteins.135 On the

inorganic side, silica based reversed-phase monoliths (Merck

Chromolith range) have been coated with cationic and anionic

ion exchangers and applied to rapid anion and cation

separations, albeit in relatively simple sample matrices.136–139

The production of a covalently bonded IDA silica monolith

for the determination of alkaline earth cations in high ionic

strength samples has recently been reported by Sugrue

et al .140,141

This work combined the selectivity of the chelating

IDA group with selective post-column reaction detection,

and, with the aid of elevated flow rates achievable through

the use of the monolithic silica support, was able to determine

sub-mg L21 concentrations of calcium and magnesium in 2 M

KCl and NaCl brines in 40 s. Fig. 6 shows this impressive

separation and illustrates the future potential such phases

have for rapid and even on-line analysis of complex matrices.

Rybalko and Nesterenko142 have also demonstrated an

interesting application of a linear flow gradient to the

separation of inorganic anions on 3 mm CIM-disk function-

alised with L-hydroxyproline.

5.2 New phases for IC-MS

The recent combination of IC with MS detection is ideally

suited to complex sample analysis and so also deserves specific

mention. To facilitate the coupling of the two techniques, new

low capacity stationary phases are required which can be used

with MS compatible eluents. Suppression of hydroxide eluents

is the approach being promoted by Dionex, who now supply

several hydroxide selective phases specifically packed in micro-

bore columns for use with MS detection. The application of

IC-MS to the trace ion analysis of a number of complex

sample matrices has already been shown, including trace

oxyhalides in various water samples,143,144 ionic species within

agricultural chemicals145 and a range of other ionic contami-

nants, including ultra-trace perchlorate in environmental water

samples.146,156

6 Conclusions

It is clear from this review that over the past ten years IC has

moved into whole new areas of complex application, and a

major part of this is due to developments into new and more

selective stationary phases. Much of this progression has been

gradual, with small refinements in selectivity and efficiency

being tailor made for specific complex applications. This will

of course continue on apace, and it is clear that at this point in

time, and for the immediate future, IC holds a dominant

position in the field of ion analysis. However, analytical

techniques need to evolve or they will soon be superseded. The

movement of IC into the field of bio-analysis over the past few

years has seen a substantial increase in new and challenging

applications. It is likely that increasing numbers of novelcolumns and stationary phases will be developed to tackle

these new and complex problems and this alone means

research into the development and application of IC is still

an exciting place to be.

Brett Paull*a and Pavel N. Nesterenkob

aNational Centre for Sensor Research, School of Chemical Sciences,Dublin City University, Glasnevin, Dublin 9, Ireland.E-mail: [email protected]; Fax: +353 (0)1 7005503;Tel: +353 (0)1 7005060bDepartment of Analytical Chemistry, Moscow State University,Moscow, 119899, Russian Federation.E-mail: [email protected]; Fax: +7 (095) 939 46 75;Tel: +7 (095) 939 44 16

Fig. 6 Shows the separation of 10 mg L21 Mg(II) and 10 mg L21

Ca(II) in 2 M KCl (15% w/w) solution in under 40 s. Column 5 10 cm

IDA silica monolith. Eluent 5 1 M KCl, pH 4.85, flow rate 5

5 mL min21. Peaks; 1 5 Mg(II), 2 5 Ca(II). From ref. 140—

Reproduced by permission of The Royal Society of Chemistry.




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Novel Ion Chromatographic Stationary Phases for the Analysis of Complex

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